Phosphofructokinase (PFK) is the rate-limiting enzyme for glycolysis, although its regulation is poorly understood. The understanding of PFK regulation may be of particular importance in abnormal states such as diabetes and insulin resistance, since under physiological conditions (i.e. plasma insulin less than 100 uU/ml) a substantial portion of a glucose load (orally ingested or infused) is oxidized by skeletal muscle. To increase the oxidation of glucose, glycolysis must be accelerated. Glucose 1,6- bisphosphate (GP2) is a potent activator of PFK in vitro and could thus be a potentially important regulator of PFK in vivo. We have studied a number of regulators of PFK underconditions of anoxia, isometric contraction, and during euglycemic, hyperinsulinemia, in human skeletal muscle. Biopsies were obtained from the quadriceps femoris muscle with the needle biopsy technique. During anoxia, increases in ADP, AMP, Pi, fructose 1,6- bisphosphate (FP2), and F6P and decreases in phosphocreatine (PCr) are responsible for activation of glycolysis. During isometric contraction, increases in GP2 were observed in all subjects and the increase in GP2 was well correlated with the in vivo glycolytic rate. During euglycemic-hyperinsulinemia in insulin-sensitive men, GP2 increased markedly in all subjects. However, there were no changes in any of the other measured modulators of PFK (i.e. PCr, ATP, ADP, AMP, FP2, F6P, and citrate). Hence it is possible that adequate increases in GP2 are a prerequisite for insulin-mediated glucose disposal in skeletal muscle. We are currently investigating the changes in PFK regulators in subjects with impaired glucose tolerance.